The most abundant protein component in circulating blood of mammalian species is serum albumin, which is normally present at a concentration of approximately 3 to 4.5 grams per 100 milliters of whole blood. Serum albumin is a blood protein of approximately 70,000 daltons which provides several important functions in the circulatory system. For instance, it functions as a transporter of a variety of organic molecules found in the blood, as the main transporter of various metabolites such as fatty acids and bilirubin through the blood, and, owing to its abundance, as an osmotic regulator of the circulating blood. Human serum albumin (HSA) has been used clinically in protein replacement therapy and as a plasma expander for patients that have experienced blood loss, e.g., resulting from surgery, burns, trauma, or shock.
Since patients often receive large quantities of HSA in a single treatment, commercial HSA must have a higher degree of purity than many other proteins used therapeutically. The protein must also have the correct conformation to avoid antigenic responses.
HSA is obtained in useful quantities either by purification from human serum derived from human blood donors or by expression and isolation from a recombinant expression system, e.g., transgenic murine milk (Shani et al., Tranasgenic Res., 1: 195–208 (1992)), Pichia pastoris (Kobayashi et al., Ther. Apher, 2: 257–262 (1998)), and transgenic leafy or tuber plants, such as tobacco and potato plants (Sijmons et al., Biotechnology (NY), 8: 217–221 (1990)). Since HSA harvested from human serum must be purified away from any possible human pathogens and then scrupulously tested, recombinant sources have a tremendous advantage in that they lack such transmissible pathogens.
In research and assay protocols, serum albumin has found a variety of uses. For example, serum albumin is used as a component in various tissue culture growth media to grow eukaryotic, and especially mammalian, cells. Serum albumin may also be used as a blocking protein in various assay protocols, such as in enzyme-linked immunosorbent assays (ELISAs) and Western immunoblots, to prevent potential interference due to non-specific binding by other molecules. In addition, serum albumin may also be used as a carrier molecule to which antigens may be adsorbed or conjugated to form immunogenic compounds, which elicit antibody production to the particular antigen. The size of serum albumin also makes it useful as a standard molecular weight marker protein, which may be used to estimate or calculate the size of other proteins by comparison.
Clearly, serum albumin is a protein that has found and will continue to find use in a wide variety of medicinal, diagnostic, and research applications. Of particular importance is the demand for highly purified serum albumin, especially highly purified HSA. Typically, methods of obtaining highly purified preparations of HSA include a step that uses affinity chromatography with a dye conjugated to a matrix or resin, such as Cibacron Blue SEPHAROSE® affinity matrix (Amersham Pharmacia Biotech, Upsala, Sweden). However, current dye-based affinity chromatography is not able to provide highly purified HSA in a single step and, therefore, requires additional steps that increase production time and costs.
Accordingly, there is a continuing need for the means and methods for producing serum albumins, and especially HSA, in a highly purified state and in greater yield using fewer production steps. In addition, needs remain for means and methods to more thoroughly remove or trap serum albumins from a solution, including whole blood, in various processes and production methods.